Micro-incubation systems for microfluidic cell culture and methods

ABSTRACT

A micro-incubator manifold for improved microfluidic configurations and systems and methods of manufacture and operation for a manifold and automated microfluidic systems.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from provisional patentapplication 61/566,651, filed Dec. 3, 2011, which is hereby incorporatedby reference for all purposes.

This application incorporates each of the following provisional patentapplications by reference:

61/367,371 filed Jul. 23, 2010

61/297,278 filed Jan. 21, 2010

61/471,103 filed Apr. 1, 2011

This application is related to material discussed in one or more of thefollowing applications, each of which are incorporated herein byreference for all purposes: U.S. Provisional Patent Application No.61/037,297, filed Mar. 17, 2008; U.S. Provisional Patent Application No.61/018,882, filed Jan. 3, 2008; U.S. patent application Ser. No.11/994,997, filed Aug. 11, 2008, which is a National Stage Entry ofPCT/US06/26364, filed Jul. 6, 2006 and which claims priority from U.S.Provisional Patent Application No. 60/773,467, filed Feb. 14, 2006 andfrom U.S. Provisional Patent Application No. 60/697,449, filed Jul. 7,2005; U.S. patent application Ser. No. 12/019,857, filed Jan. 25, 2008,which claims priority to U.S. Provisional Patent Application No.60/900,651 filed Feb. 8, 2007; U.S. patent application Ser. No.11/648,207, filed Dec. 29, 2006, now issued as U.S. Pat. No. 8,257,964,which claims priority to U.S. Provisional Patent Application No.60/756,399, filed Jan. 4, 2006; and U.S. patent application Ser. No.12/348,907, filed Jan. 5, 2009.

COPYRIGHT NOTICE

Pursuant to 37 C.F.R. 1.71(e), applicants note that a portion of thisdisclosure contains material that is subject to copyright protection(such as, but not limited to, diagrams, device photographs, or any otheraspects of this submission for which copyright protection is or may beavailable in any jurisdiction). The copyright owner has no objection tothe facsimile reproduction by anyone of the patent document or patentdisclosure, as it appears in the Patent and Trademark Office patent fileor records, but otherwise reserves all copyright rights whatsoever.

FIELD OF THE INVENTION

The invention in various embodiments relates to assays, systems, and/ordevices for culturing cells or other biologic material in controlledenvironments and that are applicable to related fields generally usingmicrofluidic systems. Particular embodiments involve configurations thatcan be used with various standard automated handling systems, withactive or passive loading and perfusion of medium and to providehigh-throughput multi-assay automated systems for culturing, viewing,and analyzing cell growth, invasion, movement, chemotaxis or otherproperties. More specifically, the invention relates in specificembodiments to heat control systems for microfluidic culture plates andother automated systems for culture plates.

BACKGROUND OF THE INVENTION

The discussion of any work, publications, sales, or activity anywhere inthis submission, including in any documents submitted with thisapplication, shall not be taken as an admission that any such workconstitutes prior art. The discussion of any activity, work, orpublication herein is not an admission that such activity, work, orpublication existed or was known in any particular jurisdiction.

Microfluidic cell culture is an important technology for applications indrug screening, tissue culturing, toxicity screening, and biologicresearch and can provide improved biological function, higher-qualitycell-based data, reduced reagent consumption, and lower cost. Highquality molecular and cellular sample preparations are important forvarious clinical, research, and other applications. In vitro samplesthat closely represent their in vivo characteristics can potentiallybenefit a wide range of molecular and cellular applications. Handling,characterization, culturing, and visualization of cells or otherbiologically or chemically active materials (such as beads coated withvarious biological molecules) has become increasingly valued in thefields of drug discovery, disease diagnoses and analysis, and a varietyof other therapeutic and experimental work.

Numerous aspects related to microfluidic systems, devices, methods andmanufacturing are discussed in the above-referenced and related patentapplications. While no particular limitations should be read form thoseapplications into any claims presented herein, these incorporateddocuments provide useful background material related to specificembodiments.

Other publications and/or patent documents that discuss variousstrategies related to cell culture using microfluidic systems andrelated activities include the following U.S. patent applications andnon-patent literature, which, along with all citations therein, areincorporated herein by reference for all purposes. A listing of thesereferences here does not indicate the references constitute prior art.

Cytoplex, Inc. U.S. Pat. No. 6,653,124 “Array-based microenvironment forcell culturing, cell monitoring and drug-target validation.”

Cellomics, Inc. U.S. Pat. No. 6,548,263 “Miniaturized cell array methodsand apparatus for cell-based screening.”

Fluidigm, Inc. Published Application 20040229349 (Nov. 18, 2004)“Microfluidic particle-analysis systems.”

Earlier work and patent applications as cited above, involving at leastone of the present inventors, discuss various configurations, methods,and systems related to microfluidic cell culture and that work and thosepublications are is incorporated herein by reference.

SUMMARY

The present invention involves various components, systems, and methodsrelated to improved microfluidic cell culture devices and systems, inparticular systems for the culturing and/or analysis and/or viewing ofcells under controlled temperature and gas parameters. In one aspect,the invention involves novel microfluidic cell culture devices, systemsand methods that have advantages over previously proposed culturesystems providing in some embodiments, more convenient and unobtrusiveheating and gas control and also providing automatic handling. Inanother aspect, the invention involves novel structures and methods forintegrating heating and gas control with multiple microfluidic cellculture units in various multi cell culture unit systems, such as to amicrotiter well plate structure including various standard well plateformats (e.g., a 96-well SBS culture plate, or other plate formats,including plates having 6, 12, 24, 96, 384 or 1536 sample wells, as wellas open bottom standard well plates, as well as plates with specificareas for cell culture and/or viewing.

Removable Multi-Chamber Plate Manifold with Gas and/or TemperatureControl

In particular embodiments and examples, design features includeproviding a cell culture device in a convenient format that allows forthe elimination of tubing and connectors to the plates themselves andprovides temperature control and monitoring mostly or entirely containedin a removable manifold that fits onto the plates with a gas seal,thereby providing the ability to maintain long-term cell culture withtemperature and/or gas control in a culture platform that maintains theability to easily observe cells and that is easily removable from theculture plate. A system of the invention can be used with a variety ofcell culture units on culture plates, such as those described in theabove referenced patent applications, including cell culture units fordetermining cellular invasion, culture units with gel culture media, anda variety of other culture units as described herein or in incorporatedrelated applications.

Living cells generally require careful control of their physicalenvironment, including nutrient and gas exchange, temperatureregulation, and protection from stress. Advanced micro-scale cellculture methods such as described in above referenced patentapplications enable structural control (microfabrication), and perfusioncontrol (microfluidics).

Micro-Incubation, and Micro-Incubator Chamber/Well in ContactMicrofluidic Cell Culture Chambers

According to specific embodiments, the present invention is directed toan additional area of this field, referred to at times herein asmicro-incubation, to provide control of temperature and gas atmospherefor use in micro-scale cell culture systems in a way that is unobtrusiveto observational equipment and that further is easily attached andremoved from a culture plate in specific embodiments.

According to specific embodiments, the invention provides aminiaturization of the traditional cell culture incubator concept toperform dynamic, continuous temperature and gas regulation directly to amicrofluidic chamber. In example implementations, this is possiblethrough the creation of a micro-incubator chamber of about 5 mL volumein contact with the microfluidic cell culture chambers (1 ul). Thetemperature and gas content of the micro-incubator quickly transfers tothe cell culture chamber (by conduction and diffusion). In specificembodiments, the micro-incubator is maintained by a novel manifolddesign as described herein. Manifolds according to specific embodimentsof the invention can be controlled by various systems and software.

System for Time-Lapse Microscopy of Living Cells

Many products and methods enable time-lapse microscopy of living cells.Three approaches that are commonly known can generally be understood as:(1) full microscope enclosures, (2) stage-top incubators, and (3)perfusion chambers.

Full microscope enclosures surround the entire microscope except forsome heat generating or heat sensitive components such as the camera andillumination sources. The air within the enclosure is circulated andmaintained at the desired temperature and gas environment of the sample.An advantage of this method is that temperature control of the wholemicroscope greatly reduces focus drifts due to room temperaturefluctuations, but numerous drawbacks include expensive and customizedconstruction, obstruction of access to microscope, and high consumptionof energy and gases. Also, exposure to humidity and repeated temperaturechanges may damage microscopes.

Stagetop incubators surround only a small volume intended to house oneor more Petri dishes, slides or other culture platform. These providelocal temperature regulation and enable limited gas environment control.They are convenient, but do not provide the same level of control as amicroscope enclosure, as the stagetop incubator must mimic the mechanicsof an enclosure, but in a smaller size and adapt to the microscope. Forexample, uniform temperature control in a stagetop incubator is limitedby the heat sink of the stage itself, and cutouts to provide for thelight path for optical clarity further reduce uniformity. In addition,the complexity of the design of stagetop incubators make them expensive.

Perfusion chambers generally consist of an assembly that encloses aflowing liquid volume, with heating elements either directly through thewalls of the chamber or immediately upstream of the inlet flow path. Thedesign of the control elements need to be carefully considered, asissues such as heat/mass transfer may make proper maintenance of asteady state condition difficult. At present, flow chambers areinfrequently used in live cell imaging due to the myriad difficulties ofadapting them for typical uses.

The present invention, by contrast, integrates temperature, flow, andgas control directly to a microfluidic culture plate via the use of amanifold that seals to the microfluidic plate. While manifolds similarin some aspects are discussed in some of the above referencedapplications to control perfusion on microfluidic plates, the previousdesigns did not incorporate all of the features described herein as suchincorporation was difficult due to the compact nature of the manifoldand culture plate. The present manifold design includes noveltemperature or gas “microincubator” compartments created by operation ofthe manifold. These compartments have been demonstrated to provide noveland critical advantages for the proper integration for live cell imagingneeds.

The present invention enables direct cell culture on a microscope stagewithout the use of external environment chambers such as enclosures orstage-top heaters. The “microincubator” concept according to specificembodiments of the present invention provides precise control, long-termcell culture, and ease of dynamic changes of conditions that has notbeen available in this context in other designs.

While many of the examples discussed in detail herein are designed to beused in conjunction with a standard or custom well plate, according tospecific embodiments the microfluidic structures and culture units andsystems and methods of various configurations as described herein canalso be deployed independently of any well-plate, such as in variousintegrated lab-on-a-chip systems that are not configured to be used inconjunction with well plates or various other microfluidic devices orsystems.

For purposes of clarity, this discussion refers to devices, methods, andconcepts in terms of specific examples. However, the invention andaspects thereof may have applications to a variety of types of devicesand systems. It is therefore intended that the invention not be limitedexcept as provided in the attached claims and equivalents.

Furthermore, it is well known in the art that systems and methods suchas described herein can include a variety of different components anddifferent functions in a modular fashion. Different embodiments of theinvention can include different mixtures of elements and functions andmay group various functions as parts of various elements. For purposesof clarity, the invention is described in terms of systems that includemany different innovative components and innovative combinations ofinnovative components and known components. No inference should be takento limit the invention to combinations containing all of the innovativecomponents listed in any illustrative embodiment in this specification.Unless specifically stated otherwise herein, any combination of elementsdescribed herein should be understood to include every sub-combinationof any subset of those elements and also any sub-combination of anysubset of those elements combined with any other element describedherein or as would be understood to a practitioner of skill in the art.

In some of the drawings and detailed descriptions below, the presentinvention is described in terms of the important independent embodimentsof multi-component devices or systems. This should not be taken to limitvarious novel aspects of the invention, which, using the teachingsprovided herein, can be applied to a number of other situations. In someof the drawings and descriptions below, the present invention isdescribed in terms of a number of specific example embodiments includingspecific parameters related to dimensions of structures, pressures orvolumes of liquids, temperatures, electrical values, durations of time,and the like. Except where so provided in the attached claims, theseparameters are provided as examples and do not limit the invention,which encompasses other devices or systems with different dimensions.For purposes of providing a more illuminating description, particularknown fabrication steps, cell handling steps, reagents, chemical ormechanical process, and other known components that may be included tomake a system or manufacture a device according to specific embodimentsof the invention are given as examples. It will be understood to thoseof skill in the art that except were specifically noted hereinotherwise, various known substitutions can be made in the processesdescribed herein.

All references, publications, patents, and patent applications cited inthis submission are hereby incorporated by reference in their entiretyfor all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a side view of an example micro-incubator manifoldaccording to specific embodiments of the invention shown in place in amicro-incubator system with a well plate and microscope viewer.

FIG. 2 illustrates one example of a top plane view of a manifold with aheat controller according to specific embodiments of the invention.

FIG. 3 illustrates one example of a topside view of a manifold with aheat controller sealed to a well-plate and mounted in a microscopeviewer according to specific embodiments of the invention.

FIG. 4 illustrates one example of a culture plate with four cultureunits placed onto a 96-well standard SBS plate. This example shows fourculture units (corresponding to rows labeled A-D) with six inlet wells(labeled A1-D6), four microfluidic culture areas placed under the largeviewing oval, and two outlet wells (7-8). This is an example only andplacement and designation of the various wells and components will varywith different implementations.

FIG. 5A-D are schematic drawings of an example implementation of amanifold with a heat controller from various views according to specificembodiments of the invention.

FIG. 6(A) illustrates the bottom portion of a heat exchange moduleaccording to specific embodiments of the invention. The bottom portionattaches to the pneumatic portion of the manifold. (B) illustrates thetop portion of a heat exchange module according to specific embodimentsof the invention.

FIG. 7 is a schematic side view of the pneumatic portions of a manifoldsealed to a culture plate and showing the gas in lines connecting to anenvironment control volume according to specific embodiments of theinvention.

FIG. 8 is a schematic showing how a manifold interfaces to amicrofluidic plate according to specific embodiments wherein a positiveseal is created by applying a vacuum to the cavity surrounding all thewells. The heating unit is not shown in this figure.

FIG. 9 illustrates a manifold with additional gas line and a heatedobjective lens according to an earlier manifold design.

FIG. 10A-C shows a top view, side view, and plan view of a schematic ofan example pneumatic manifold according to earlier designs. In thisexample, the eight tubing lines to the right are for compressed air, andeach is configured to provide pressure to a column of cell inlet wellsin a microfluidic array. The left-most line in the figure is for vacuumand connects to an outer vacuum ring around the manifold. This basicmanifold design is modified using the teachings herein to produce theheated manifold.

FIG. 11 illustrates an example microfluidic perfusion system (ONIX™),microincubator controller and manifold (MIC) according to specificembodiments of the invention.

FIG. 12 illustrates an example microfluidic perfusion system (ONIX™),microincubator controller and manifold (MIC) and computer control systemaccording to specific embodiments of the invention.

FIG. 13 shows NIH-3T3 mouse fibroblasts cultured using themicroincubator system according to specific embodiments of the inventionat t=0 (left) and after 15 hours (right) showing cell growth andviability. When no temperature or CO₂ was controlled, the cells rapidlydied within 2 hours.

FIGS. 14A-B illustrate one alternative of plate and culture unit designwith an example culture unit filled with blue dye with the image takenfrom top according to specific embodiments of the invention.

FIG. 15 is a screenshot showing integration of the ONIX microfluidicperfusion system with an open-source microscopy application.

FIG. 16 illustrates a microincubation system integrated with amicroscope system for cell analysis. The dimensions of the manifold inspecific embodiments allow it to sit on a standard well plate stage,with a transparent optical path that does not interfere with lightmicroscopy. This allows time-lapsed imaging of cells cultured in themicro-incubator.

FIG. 17 is a block diagram showing a representative example logic devicein which various aspects of the present invention may be embodied.

FIG. 18A is a block diagram showing an automated piston driven systemaccording to specific embodiments of the invention. FIG. 18B illustratesperspective views of the automated piston driven system of FIG. 18A inthe elected and sealed states.

FIG. 19 (Table 1) illustrates an example of diseases, conditions, orstates that can be evaluated or for which drugs or other therapies canbe tested according to specific embodiments of the present invention.

FIG. 20A-D are schematics of an example implementation of electroniccontrol circuits for a manifold according to specific embodiments of theinvention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS 1. Overview Definitions

A “particle” refers to biological cells, such as mammalian or bacterialcells, viral particles, or liposomal or other particles that may besubject to assay in accordance with the invention. Such particles haveminimum dimensions between about 50-100 nm, and may be as large as 20microns or more. When used to describe a cell assay in accordance withthe invention, the terms “particles” and “cells” may be usedinterchangeably.

A “microchannel” or “channel” or “flow channel” generally refers to amicron-scale channel used for fluidically connecting various componentsof systems and devices according to specific embodiments of theinvention. A microchannel typically has a rectangular, e.g., square, orrounded cross-section, with side and depth dimensions in a preferredembodiment of between 10 and 500 microns, and 10 and 500 microns,respectively. Fluids flowing in the microchannels may exhibitmicrofluidic behavior. When used to refer to a microchannel within themicrowell array device of the invention, the term “microchannel” and“channel” are used interchangeably. “Flow channel” generally denoteschannels designed for passage of media, reagents, or other fluids orgels and in some embodiments cells. “Culture channel” or “cell culturechannel” generally denotes a portion of a cell culture structure thatcells are designed to flow through and also remain during cell culture(though the cells may be localized into a particular culture area of theculture channel in some embodiments). “Air channel” generally denotes aroughly micron-scale channel used for allowing gases, such as air,oxygen enriched mixtures, etc., to pass in proximity to flow channels orculture areas. “Perfusion channel” is sometimes used to indicate a flowchannel and any perfusion passages or structures that allow media toperfuse to the culture area.

A “perfusion barrier” refers to a combination of solid structures andperfusion passages that generally separate a flow channel from a cellculture area or chamber. The perfusion passages are generally smallerthan the microchannel height and/or width (for example, on the order of5-50% or on the order of about 10%) and are designed to keep cells,other culture items, and in some embodiments gels, from migrating intothe flow channels, while allowing some fluidic flow that is generally ofa much higher fluidic resistance than the fluid flow in the flowchannels. In one example embodiment, the perfusion barrier has aperfusion passage that is 4 microns high and that otherwise runs most ofthe length of the microchannel. In other embodiments, a perfusionbarrier has many perfusion passages that are about as high as themicrofluidic channel, but about 4 microns wide.

A “microfluidics device” refers to a device having various station orwells connected by micron-scale microchannels in which fluids willexhibit microfluidic behavior in their flow through the channels.

A “microwell array” refers to an array of two or more microwells formedon a substrate.

A “device” is a term widely used in the art and encompasses a broadrange of meaning. For example, at its most basic and least elaboratedlevel, “device” may signify simply a substrate with features such aschannels, chambers and ports. At increasing levels of elaboration, the“device” may further comprise a substrate enclosing said features, orother layers having microfluidic features that operate in concert orindependently. At its most elaborated level, the “device” may comprise afully functional substrate mated with an object that facilitatesinteraction between the external world and the microfluidic features ofthe substrate. Such an object may variously be termed a holder,enclosure, housing, or similar term, as discussed below. As used herein,the term “device” refers to any of these embodiments or levels ofelaboration that the context may indicate.

Microfluidic systems provide a powerful tool to conduct biologicalexperiments. Recently, elastomer-based microfluidics has especiallygained popularity because of its optical transparency, gas permeabilityand simple fabrication methods. The present invention involvesintegrated microfluidics used for various culture and assay applicationsand systems for providing heating control and automating varioushandling of culture plates. Advantages of specific embodiments includeuse of a standard-sized microtiter plate format, tubing free plates, andeasy and effective mating of plates with a manifold to provide gasrecirculation and heating control.

According to further embodiments of the invention, as has beenpreviously described, a novel cell loading system uses a pneumaticmanifold and pneumatic pressure to place cells in the micro culturearea. With the addition of this cell loading system, microfluidic cellculture and analysis can be fully automated using other automatedequipment that exists for handling standard titer plates. In the presentinvention, heating and gas circulation elements are incorporated intothe manifold to provide a micro-incubator system.

In further embodiments, a gravity driven flow culture configurationutilizes the medium level difference between the inlet and outlet wellas well as engineering the fluidic resistances to achieve the desirableflow rate in nL/min regime can be used to “passively” flow culturemedium for long periods of time (e.g., up to 4 days) without the use ofbulky external pumps or tubes in an environment such as an incubator tocontrol temperature and then the heat controlled manifold, as providedherein, can be used for control of the cell culture during observation.

In some embodiments, a custom pneumatic flow controller can be attachedto the gas and electric connectors in the manifold and thereby used toload the cells into the culture regions, to switch between differentexposure solutions, and to control the temperature of the cultureregion. A digital software interface can be used to allow a user toprogram specific inputs (pulses, ramps, etc.) over time to expose thecells to complex functions during time-lapse imaging while maintainingor varying temperature and gas exposure as desired.

2. Microfluidic Culture System and Array

The applications referenced above discussed a variety of different cellculture configurations and fabrication techniques. Portions of theoperation of the cell culture areas and materials are useful asbackground to the present discussion. In some examples therein, one ormore micro culture areas are connected to a medium or reagent channelvia a grid of fluidic passages (or diffusion inlets or conduits),wherein the grid comprises a plurality of intersecting high fluidicresistance perfusion passages. In one discussed example, passages in thegrid are about 1 to 4 μm in height, 25 to 50 μm in length and 5 to 10 μmin width, the grid allowing for more even diffusion between medium orreagent channels and the culture area and allowing for easiermanufacturing and more even diffusion. The earlier application furtherdiscussed that the high fluidic resistance ratio between themicrochamber and the perfusion/diffusion passages or grid (e.g., ratiosin the range of about 10:1, 20:1 to 30:1) offers many advantages forcell culture such as: (1) size exclusion of cells; (2) localization ofcells inside a microchamber; (3) promoting a uniform fluidic environmentfor cell growth; (4) ability to configure arrays of microchambers orculture areas; (4) ease of fabrication, and (5) manipulation of reagentswithout an extensive valve network. Examples were illustrated wherein agrid-like perfusion barrier can be much shorter than the culture area orcan be near to or at the same height, according to specific embodimentsof the invention and further wherein various configurations for culturedevices were illustrated.

3. Pneumatic Manifold with Heat Control

As discussed above, one difficulty in a number of culture systems is howto control the heating and temperature of the culture area whileallowing for observation of the cellular processes. Previous solutionshave relied on heating sources applied to the well-plate, for example,from the microscope viewer. (E.g., see FIG. 11), or containing theentire system in a controlled environment.

The present invention provides an improved culture system by placing allor nearly all heat and gas controls in or attached to a typicallyremovable manifold that in preferred embodiments is configured to beoperational will not interfering with observational equipment. Theinvention will be more easily understood with reference to the specificexamples illustrated, though these examples are intended to beillustrative and not limiting. FIG. 1 shows a side view of an examplemicro-incubator manifold according to specific embodiments of theinvention shown in place in a micro-incubator system with a well plateand microscope viewer. FIG. 2 illustrates one example of a top planeview of a manifold with a heat controller according to specificembodiments of the invention. FIG. 3 illustrates one example of atopside view of a manifold with a heat controller sealed to a well-plateand mounted in a microscope viewer according to specific embodiments ofthe invention. FIG. 4 illustrates one example of a culture plate withfour culture units placed onto a 96-well standard SBS plate. Thisexample shows four culture units (corresponding to rows labeled A-D)with six inlet wells (labeled A1-D6), four microfluidic culture areasplaced under the large viewing oval, and two outlet wells (7-8).

Thus, according to specific embodiments, a MicroIncubator Manifold caninterface to a variety of microfluidic plates and provides one or moreof cell-loading, perfusion, temperature and gas environment control. Aconvective heat exchanger adds or removes heat to the gas mixture usinga Peltier thermoelectric device. The cells are kept warm throughconducted heat from the warm gas mixture and the desired gasconcentration diffuses in the media surrounding the cells through a gaspermeable membrane on the microfluidic plate.

As can be seen from FIG. 1, when the manifold according to specificembodiments is in place over a culture plate, a sealed gas chamber isformed from the heat exchanger, under the manifold and into the areaabove the culture microfluidics. Gas introduced into the area above themicrofluidics is circulated by a fan or other gas circulatory means inthe heat exchanger, thereby providing control of both the gasenvironment and the temperature above the culture area using themanifold.

This design, according to specific embodiments of the invention, solvesthe problem of providing effective heating in a small space to deliverthe controlled temperature to the cells themselves while alsocontrolling the gas composition. According to specific embodiments ofthe invention, the microincubator manifold includes both a gas input anda recirculating fan to control the gas composition.

In the example implementation shown in FIG. 1, controlled andpressurized gases enter the manifold through the gas lines 5. Thepneumatic portion of the manifold is shown for convenience in thisexample in two pieces, top piece 10 a and bottom piece 10 b, whichcomprises a gasket for tightly fitting to plate 20, the plate containinga number of wells 22. The pneumatic operation of the manifold andfitting to the well plate is as described in herein referenced patentapplications. The manifold also includes heat exchange module 40, withinternal heat exchange fins 42 and a transparent cover plate (e.g.,glass or acrylic) 44. When the manifold is placed over plate 10, theopen region above the culture areas is connected with the heatingelement to create a gas circulation region 30. To show the device incontext, microscope and lighting elements 102 and 104 are illustrated asthey would generally be used in operation. The well plate can be anystandard or custom well plate as described herein. It will be understoodfrom the teachings provided herein that different configurations ofmanifold 40 are constructed to accommodate different well plate designs.

Recirculating on the Manifold.

In this example design, the gas and heating controls and elements areentirely contained in the manifold and the manifold can mate with anynumber of differently configured micro-well plates, including microwellplates that have no specific modifications to allow them to receive aheat source.

According to specific embodiments, two fans are used in the heatexchange element one to circulate the sealed gas in the gas area and oneto interact with the heat exchanger.

FIG. 5A-D are schematic drawings of an example implementation of amanifold with a heat controller from various views according to specificembodiments of the invention. The pneumatic portions of the manifoldoperate similarly to previously disclosed designs. The heat exchangemodule is described in more detail below.

Manifold Heat Exchange Module

A heat exchange module in one example embodiment controls thetemperature within the chamber by the cycling of air at desiredtemperature. In specific embodiments, temperature control is provided bya thermoelectric Peltier module, which are well known devices thatconvert an electric current into a temperature gradient and can also beused as a temperature controller that either heats or cools. While otherheating sources can be used in a manifold of the invention, a Peltiermodule is a presently preferred mechanism as it can be fullyincorporated into the heat exchange module and the manifold.

A heat exchange module in an example implementation has three main outerpieces: (1) a metal top enclosure, (2) a plastic bottom enclosure and(3) a manifold with an oval cutout that allows air to flow into the cellculturing chamber of the plate and cycle back out.

Plastic Bottom Enclosure

FIG. 6(A) illustrates the bottom portion of a heat exchange moduleaccording to specific embodiments of the invention. The bottom portionattaches to the pneumatic portion of the manifold. (B) illustrates thetop portion of a heat exchange module according to specific embodimentsof the invention. In a specific embodiment, the plastic enclosure of thebottom portion is attached to the top of the manifold (or example byscrews or glue or other means). The bottom of this enclosure has a cutout of two 2 mm deep flow paths. These paths merge into one over theimaging chamber. When they merge, the depth of the path rises to 3 mm.Around the outskirt of the flow path is an o-ring that prevents theexchange of air between the flow path and the environment. The size ofthe flow paths must be wide enough to allow a desirable amount flow tocirculate into the cell culture chamber. This decreases the differencein temperature in the plate cell culturing region and the Heat ExchangeModule (at the location of the temperature probe).

A vertical extrusion sits above the paths. It contains 3 chambers. Aboveone flow path is a heat sink and above the other sits a fan. The thirdchamber connects to another chamber in the metal top enclosure to makeroom for a PCB board that connects the wires of the Peltier module, asmall temperature probe, a relay, connections to the microincubatorcontroller and 2 thermal cutoffs (one for each heat sink). Thetemperature probe is routed from the electronics chamber through a screwhole on the fan and into the top of its flow path to measure thetemperature of air cycling through.

The plastic enclosure also has a cut out from the top that forms a framefor a piece of 2 mm glass. This piece of glass sits right above the cellculturing chamber in order to create optimal condition for microscopeimaging without disrupting heating.

Metal Top Enclosure

The metal top enclosure (FIG. 6B) has fin extrusion features that allowit to act as a heat sink for the other side of the Peltier module. Inaddition, it optionally contains a chamber for a second fan to speed upthe cooling process as well as room for a thermal cutoff that connectsto the electronics chamber of the plastic bottom enclosure.

After the fan is placed inside the enclosure and the thermal cutoffattached to the smaller chamber, thermal grease is applied to the top ofthe Peltier module in order to attach it to the metal top enclosure. Ina specific example, the bottom of the plastic enclosure is securelyfastened to the top enclosure, for example using screws or glue.

When temperature in the cell culturing region has to be raised, thePeltier module heats up the bottom heat sink by cooling the top heatsink. The bottom fan blows hot air across the bottom heat sink into theflow path beneath it. The heated air enters the cell culturing chamberas cooler air is circulated back to the fan. When temperature in theculturing region has to be lowered, the Peltier module cools the bottomheat sink by raising temperature on the top (up to ambient temperature).

FIG. 7 is a schematic side view of the pneumatic portions of a manifoldsealed to a culture plate and showing the gas in lines connecting to anenvironment control volume according to specific embodiments of theinvention.

FIG. 8 is a schematic showing how a manifold interfaces to amicrofluidic plate according to specific embodiments wherein a positiveseal is created by applying a vacuum to the cavity surrounding all thewells. The heating unit is not shown in this figure.

4. Earlier Pneumatic Manifold

While gravity or passive loading is effective for some microfluidic cellculture devices and desirable in some embodiments, a proprietarypneumatic manifold was previously described in the above referencedapplications. This may be mated to the plate and pneumatic pressure isapplied to the cell inlet area for cell loading and for culturing duringinvasion assays. FIG. 10A-C shows a top view, side view, and plan viewof a schematic of an example pneumatic manifold according to earlierdesigns. In this example, the eight tubing lines to the right are forcompressed air, and each is configured to provide pressure to a columnof cell inlet wells in a microfluidic array. The left-most line in thefigure is for vacuum and connects to an outer vacuum ring around themanifold. The manifold is placed on top of a standard well plate. Arubber gasket lies between the plate and manifold, with holes matchingthe manifold (not shown). The vacuum line creates a vacuum in thecavities between the wells, holding the plate and manifold together.Pressure is applied to the wells to drive liquid into the microfluidicchannels (not shown). A typical pressure of 1 psi is used; therefore thevacuum strength is sufficient to maintain an air-tight seal. In oneexample there are 9 tubing lines to the pressure controller: 8 lines arefor compressed air and 1 line is for vacuum (leftmost). In specificexample embodiments, each column is connected to a single pressure line.Columns above the cell imaging regions are skipped.

Pressurized cell loading has been found to be particularly effective inpreparing cultures of aggregating cells (e.g., solid tumor, liver,muscle, etc.). Pressurized cell loading also allows structures withelongated culture regions to be effectively loaded. Use of a pressurizedmanifold for cell loading and passive flow for perfusion operationsallows the invention to utilize a fairly simple two inlet design,without the need for additional inlet wells and/or valves as used inother designs.

While this manifold is effective for cell loading and some perfusiontasks, the manifold did not effectively provide for the recirculation ofa gas over the culture area or for any heat control. As illustrated inthe figure, heating was provided when necessary from the opposite sideof the culture plate, for example form the vicinity of the microscopeviewer.

The plate manifold optionally also included an additional “gas line”that is used to bathe the cells in the microfluidic device with aspecified gas environment (for example, 5% CO₂). Other examples includeoxygen and nitrogen control, but any gaseous mixture can be sent to thecells. The gas flows through the manifold into the sealed wells abovethe cell culture area and holes in the microfluidic device enable thegas to flow into specified microfluidic air channels, as describedabove. The gas permeable device layer (PDMS) allows the gas to diffuseinto the culture medium prior to exposing the cells. By continuouslyflowing the gas through the microfluidic plate, a stable gas environmentis maintained. This provides an optional means for controlling the gasenvironment to placing the microfluidic plate into an incubator.

FIG. 12 illustrates an example microfluidic perfusion system (ONIX™),microincubator controller and manifold (MIC) and computer control systemaccording to specific embodiments of the invention.

As just one example, FIG. 14 illustrates one alternative of plate andculture unit design with an example culture unit filled with blue dyewith the image taken from top according to specific embodiments of theinvention. However, any culture units in any configuration of cultureplates can be used with a correctly dimensioned manifold according tospecific embodiments of the invention. This includes open top units,invasion units, liver mimetic units, gel units, etc as described inabove referenced applications.

Cell Assay and/or Observation

Cell assay can be performed directly on the microfluidic cell cultureusing standard optically based reagent kits (e.g. fluorescence,absorbance, luminescence, etc.). For example a cell viability assayutilizing conversion of a substrate to a fluorescent molecule by livecells has been demonstrated (CellTiter Blue reagent by PromegaCorporation). The reagent is dispensed into the flow inlet reservoir andexposed to the cells via gravity perfusion over a period of time (e.g.,21 hours). For faster introduction of a reagent or other fluid, the newfluid can be added to the flow inlet reservoir followed by aspiration ofthe cell inlet reservoir.

Data can be collected directly on the cells/liquid in the microfluidicplate, such as placing the plate into a standard fluorescence platereader (e.g., Biotek Instruments Synergy 2 model). In some reactions,the substrate may diffuse into the outlet medium, and therefore beeasily detected in the cell inlet reservoir. For cell imaging assays,the plate can be placed on a scanning microscope or high content system.For example, an automated Olympus IX71 inverted microscope station canbe used to capture viability of cultured liver cells with a 20×objective lens.

By repeatedly filling/aspirating the wells, cells can be maintained forlong periods of time with minimal effort (e.g. compared to standard“bioreactors” which require extensive sterile preparation of large fluidreservoirs that cannot be easily swapped out during operation).

Example Cell Culture

Cells were cultured using the micro-incubation system to controltemperature and gas atmosphere. In one example, human cancer cells(HT-1080, MCF-7, MDA-MB-231) were cultured at 37C and 5% CO2 to monitorcell division over 24 hours. Additional cell types, including yeast,bacteria, primary cells, neurons, etc. have been successfully culturedusing the microincubation system. As an example, FIG. 13 shows NIH-3T3mouse fibroblasts cultured using the microincubator system according tospecific embodiments of the invention at t=0 (left) and after 15 hours(right) showing cell growth and viability. When no temperature or CO2was controlled, the cells rapidly died within 2 hours.

Integrated Systems

Integrated systems for the collection and analysis of cellular and otherdata as well as for the compilation, storage and access of the databasesof the invention, typically include a digital computer with softwareincluding an instruction set for sequence searching and/or analysis,and, optionally, one or more of high-throughput sample control software,image analysis software, collected data interpretation software, arobotic control armature for transferring solutions from a source to adestination (such as a detection device) operably linked to the digitalcomputer, an input device (e.g., a computer keyboard) for enteringsubject data to the digital computer, or to control analysis operationsor high throughput sample transfer by the robotic control armature.Optionally, the integrated system further comprises valves,concentration gradients, fluidic multiplexors and/or other microfluidicstructures for interfacing to a microchamber as described.

Readily available computational hardware resources using standardoperating systems can be employed and modified according to theteachings provided herein, e.g., a PC (Intel x86 or Pentiumchip-compatible DOS,™ OS2,™ WINDOWS,™ WINDOWS NT,™ WINDOWS95,™WINDOWS98,™ LINUX, or even Macintosh, Sun or PCs will suffice) for usein the integrated systems of the invention. Current art in softwaretechnology is adequate to allow implementation of the methods taughtherein on a computer system. Thus, in specific embodiments, the presentinvention can comprise a set of logic instructions (either software, orhardware encoded instructions) for performing one or more of the methodsas taught herein. For example, software for providing the data and/orstatistical analysis can be constructed by one of skill using a standardprogramming language such as Visual Basic, Fortran, Basic, Java, or thelike. Such software can also be constructed utilizing a variety ofstatistical programming languages, toolkits, or libraries.

FIG. 17 shows an information appliance (or digital device) 700 that maybe understood as a logical apparatus that can read instructions frommedia 717 and/or network port 719, which can optionally be connected toserver 720 having fixed media 722. Apparatus 700 can thereafter usethose instructions to direct server or client logic, as understood inthe art, to embody aspects of the invention. One type of logicalapparatus that may embody the invention is a computer system asillustrated in 700, containing CPU 707, optional input devices 709 and711, disk drives 715 and optional monitor 705. Fixed media 717, or fixedmedia 722 over port 719, may be used to program such a system and mayrepresent a disk-type optical or magnetic media, magnetic tape, solidstate dynamic or static memory, etc. In specific embodiments, theinvention may be embodied in whole or in part as software recorded onthis fixed media. Communication port 719 may also be used to initiallyreceive instructions that are used to program such a system and mayrepresent any type of communication connection.

Various programming methods and algorithms, including genetic algorithmsand neural networks, can be used to perform aspects of the datacollection, correlation, and storage functions, as well as otherdesirable functions, as described herein. In addition, digital or analogsystems such as digital or analog computer systems can control a varietyof other functions such as the display and/or control of input andoutput files. Software for performing the electrical analysis methods ofthe invention are also included in the computer systems of theinvention.

Auto-Sealer Automated System

FIG. 18 is a block diagram showing an automated piston driven systemaccording to specific embodiments of the invention. According to furtherspecific embodiments, an auto-sealer somewhat similar to a plate readercommonly used in biotechnology with the main difference is the design ofthe system component to allow automated handling of the microfluidicplates. In this implementation, the positive seal between manifold andmicrofluidic plate is still accomplished by applying vacuum to theinterstitial areas, but the necessary initial downward force is appliedmechanically. The area above the manifold is clear to allow access by anautomated liquid hander such as the Tecan EVO. Vacuum and pressuresensors as well as plate presence and carriage position sensors allowfor intelligent software based error handling. FIG. 18B is an imagesequence showing how the automatic sealing device accepts and seals themanifold to a microfluidic plate. A single pneumatic linear actuator(e.g., a piston) provides horizontal and vertical motion. It has beenfound that this single operation allows more precise control of themanifold and plate and holds the plate in place during operation of thepneumatic manifold.

Other Embodiments

Although the present invention has been described in terms of variousspecific embodiments, it is not intended that the invention be limitedto these embodiments. Modification within the spirit of the inventionwill be apparent to those skilled in the art.

It is understood that the examples and embodiments described herein arefor illustrative purposes and that various modifications or changes inlight thereof will be suggested by the teachings herein to personsskilled in the art and are to be included within the spirit and purviewof this application and scope of the claims.

All publications, patents, and patent applications cited herein or filedwith this submission, including any references filed as part of anInformation Disclosure Statement, are incorporated by reference in theirentirety.

What is claimed:
 1. A microfluidic cell culture system comprising: atleast one culture unit configured on a culture plate comprising: amicrofluidic culture chamber; a perfusion barrier separating saidmicrofluidic culture chamber from one or more medium flow channels; aplurality of independent inlet channels in fluid communication with saidone or more medium flow channels; and an outlet reservoir in fluidcommunication with said microfluidic culture chamber; and a pneumaticmanifold; wherein the culture plate is positively sealed to saidpneumatic manifold, allowing pressure driven control of the plurality ofindependent inlet channels so that solutions can be quickly changed overthe cells or can be multiplexed or combined in the microfluidic culturechamber; wherein said pneumatic manifold further comprises a heatexchange module for providing a circulating heat controlled gas in acontained gas volume over said microfluidic culture chamber.
 2. Thesystem of claim 1 wherein said perfusion barrier is used to reduce flowpressure from said inlet channels to said microfluidic culture chamber.3. The system of claim 1 wherein said perfusion barrier is used tocontain a gel media to said microfluidic culture chamber.
 4. The systemof claim 1 wherein said perfusion barrier mostly surrounds themicrofluidic culture chamber to separate the microfluidic culturechamber from the one or more medium flow channels.
 5. The system ofclaim 1, further comprising an automatic handling system for sealing thepneumatic manifold to the microfluidic culture plate comprising: apneumatic linear actuator cylinder; one or more position sensors; twoarms for holding the manifold from the sides, without blocking a viewthrough the top of the manifold; a plate carriage and plate grippers forholding the culture plate; said system configured so that movement ofthe cylinder causes the plate carriage to move horizontally to aposition under said manifold; said system configured so that movement ofthe cylinder causes the manifold to descend onto the plate.
 6. Thesystem of claim 5 further wherein: the cylinder is attached to the armsso that the cylinder provides both horizontal and vertical motion. 7.The system of claim 5 further wherein: said positive seal between themanifold and microfluidic culture plate is accomplished by applyingvacuum through the manifold to the interstitial areas of themicrofluidic culture plate while the necessary initial downward force isapplied mechanically.
 8. The microfluidic cell culture system of claim1, further comprising a medium well in fluid communication with said oneor more medium flow channels.
 9. The microfluidic cell culture system ofclaim 8, wherein said medium well is a gravity perfusion well.
 10. Themicrofluidic culture system of claim 1, further comprising a cell inletin fluid communication with said culture chamber.
 11. The microfluidiccell culture system of claim 1, further comprising a gas circulationregion formed by the positive seal of said pneumatic manifold to saidmicrofluidic culture plate.
 12. The microfluidic cell culture system ofclaim 11 wherein said heat exchange module further comprises a fan tocirculate gas within said gas circulation region.
 13. The microfluidiccell culture system of claim 1, wherein said heat exchange modulecomprises a convective heat exchanger.
 14. The microfluidic cell culturesystem of claim 13, wherein said convective heat exchanger comprisesheat exchange fins.
 15. The microfluidic cell culture system of claim14, wherein said heat exchange module further comprises a fan tocirculate air over said heat exchange fins.
 16. The microfluidic cellculture system of claim 1, wherein said pneumatic manifold furthercomprises a gas input.